Methods for preventing and/or treating degenerative disorders of the central nervous system

ABSTRACT

The present invention provides methods for preventing and/or treating degenerative disorders of the central nervous system using 5-(fluoromethyl)piperidine-3,4-diol, 5-(chloromethyl)piperidine-3,4-diol, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or any combination of two or more thereof. In particular, the present invention provides methods for preventing and/or treating Parkinson&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/168,101, filed Apr. 9, 2009, the content of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention provides methods for preventing and/or treatingdegenerative disorders of the central nervous system using5-(fluoromethyl)piperidine-3,4-diol,5-(chloromethyl)piperidine-3,4-diol, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof, or any combination of two or morethereof. In particular, the present invention provides methods forpreventing and/or treating Parkinson's disease.

BACKGROUND OF THE INVENTION

Many degenerative disorders of the central nervous system are associatedwith pathologic aggregation of proteins or lipids. For example,synucleinopathies are a group of diseases that arise from disruption ofsynuclein protein homeostasis. In particular, alpha-synucleinaggregation is associated with pathological conditions characterized byLewy bodies, such as Parkinson's disease, dementia with Lewy bodies, andmultiple system atrophy. Likewise, alpha-synuclein fragment, non-Abetacomponent, is found in amyloid plaques of Alzheimer's disease. Recently,enhancement of glucocerebrosidase (beta-glucosidase; GCase) activity inthe brain has been shown to prevent accumulation of synuclein in thebrain (Sean Clark, Ying Sun, You-Hai Xu, Gregory Grabowski, and BrandonWustman, “A biochemical link between Gaucher and Parkinson's disease anda potential new approach to treating synucleinopathies: apharmacological chaperone for beta-glucocerebrosidase preventsaccumulation of alpha-synuclein in a Parkinson's mouse model,” Presentedat the Society for Neuroscience Annual Meeting, San Diego, Calif.,2007). Thus, agents that enhance GCase activity may provide relief forpatients at risk for developing or diagnosed with degenerative disordersof the central nervous system.

There is a need for methods to prevent and/or treat degenerativedisorders of the central nervous system that provide patients with ahigher quality of life and achieve a better clinical outcome. Inparticular, there is a need for methods to prevent and/or treatsynucleinopathies such as Parkinson's disease and Alzheimer's diseasethat provide patients with a higher quality of life and achieve a betterclinical outcome.

SUMMARY OF THE INVENTION

The present invention provides methods for preventing and/or treating adegenerative disorder of the central nervous system in a patient at riskfor developing or diagnosed with the same, which comprises administeringto the patient in need thereof an effective amount of a therapeuticagent which is 5-(fluoromethyl)piperidine-3,4-diol,5-(chloromethyl)piperidine-3,4-diol, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof, or any combination of two or morethereof. In one embodiment, the method comprises administering5-(fluoromethyl)piperidine-3,4-diol or a pharmaceutically acceptablesalt, solvate, or prodrug thereof. In one embodiment, the methodcomprises administering (3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diolor a pharmaceutically acceptable salt, solvate, or prodrug thereof. Inone embodiment, the method comprises administering(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol hydrochloride. In oneembodiment, the method comprises administering5-(chloromethyl)piperidine-3,4-diol or a pharmaceutically acceptablesalt, solvate, or prodrug thereof. In one embodiment, the methodcomprises administering (3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diolor a pharmaceutically acceptable salt, solvate, or prodrug thereof. Inone embodiment, the method comprises administering(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol. In one embodiment, thedegenerative disorder is a synucleinopathy. In one embodiment, thedegenerative disorder is characterized by Lewy bodies. In oneembodiment, the degenerative disorder is Parkinson's disease, dementiawith Lewy bodies, multiple system atrophy or Alzheimer's disease. In oneembodiment, the degenerative disorder is associated with aggregation ofat least one protein. In one embodiment, the degenerative disorder isassociated with aggregation of alpha-synuclein. In one embodiment, thedegenerative disorder is associated with aggregation of non-Abetacomponent. In one embodiment, the degenerative disorder is associatedwith accumulation of at least one glycolipid. In one embodiment, thedegenerative disorder is associated with accumulation of at least oneglycosphingolipid. In one embodiment, the degenerative disorder isassociated with accumulation of glucocerebroside. In one embodiment, thedegenerative disorder is associated with a mutation inglucocerebrosidase. In one embodiment, the method further comprisesadministering an effective amount of at least one other therapeuticagent. In one embodiment, at least one other therapeutic agent islevodopa, an anticholinergic, a catechol-O-methyl transferase inhibitor,a dopamine receptor agonist, a monoamine oxidase inhibitor, a peripheraldecarboxylase inhibitor, or an anti-inflammatory agent.

The present invention also provides methods for preventing and/ortreating Parkinson's disease in a patient at risk for developing ordiagnosed with the same, which comprises administering to the patient inneed thereof an effective amount of(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol,(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof, or any combination of twoor more thereof.

In one embodiment, the method comprises administering(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof. In one embodiment, themethod comprises administering(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol hydrochloride. In oneembodiment, the method comprises administering(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof. In one embodiment, themethod comprises administering(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol. In one embodiment, themethod comprises administering an effective amount of at least one othertherapeutic agent. In one embodiment, at least one other therapeuticagent is levodopa, an anticholinergic, a catechol-O-methyl transferaseinhibitor, a dopamine receptor agonist, a monoamine oxidase inhibitor, aperipheral decarboxylase inhibitor, or an anti-inflammatory agent.

The present invention also provides kits comprising:

-   -   a container having an effective amount of        5-(fluoromethyl)piperidine-3,4-diol,        5-(chloromethyl)piperidine-3,4-diol, or a pharmaceutically        acceptable salt, solvate, or prodrug thereof, or any combination        of two or more thereof;    -   instructions for using the same to prevent and/or treat a        degenerative disorder of the central nervous system.        In one embodiment, the degenerative disorder of the central        nervous system is Parkinson's disease. In one embodiment, the        degenerative disorder of the central nervous system is        Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the plasma levels of reference compound, IFG, andtest compound, (3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol (referredto herein as Compound A), in mice administered a single 30 mg/kg (freebase equivalent) p.o. dose of IFG-tartrate or the hydrochloride saltform of Compound A.

FIG. 1B illustrates the brain levels of reference compound, IFG, andtest compound, Compound A, in mice administered a single 30 mg/kg (freebase equivalent) p.o. dose of IFG-tartrate or the hydrochloride saltform of Compound A.

FIG. 1C illustrates the ratio of brain to plasma levels of referencecompound, IFG, and test compound, Compound A, in mice administered asingle 30 mg/kg (free base equivalent) p.o. dose of IFG-tartrate or thehydrochloride salt form of Compound A.

FIG. 2 illustrates the brain levels of reference compound, IFG, and testcompound, (3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol (referred toherein as Compound B), in mice administered a single 30 mg/kg (free baseequivalent) p.o. dose of IFG-tartrate or free-base of Compound B.

FIGS. 3A-D illustrate the level of GCase in brain, spleen, liver andlung, respectively, of C57BL/6 mice administered a 2-week dosing regimenconsisting of nine doses of (i) control vehicle; (ii) 100 mg/kg (freebase equivalent) of reference compound, IFG-tartrate; or (iii) 10 or 100mg/kg (free base equivalent) of test compound, the hydrochloride saltform of Compound A. Note that the values appearing above the bars inthese figures represent the fold increase above control. Likewise, inthese figures, the symbol “*” represents p<0.05 t-test compared tocontrol and the symbol “#” represents p<0.05 t-test compared to micetreated with reference compound, IFG-tartrate.

FIGS. 4A-D illustrate the level of GCase in brain, spleen, liver andlung, respectively, of C57BL/6 mice administered a 2-week dosing regimenconsisting of nine doses of (i) control vehicle; (ii) 100 mg/kg (freebase equivalent) of reference compound, IFG-tartrate; or (ii) 1, 3, 10,30 or 100 mg/kg (free base equivalent) of test compound, thehydrochloride salt form of Compound A. Note that the values appearingabove the bars in these figures represent the fold increase abovecontrol. Likewise, in these figures, the symbol “*” represents p<0.05t-test compared to control and the symbol “#” represents p<0.05 t-testcompared to mice treated with reference compound, IFG-tartrate.

FIGS. 5A-D illustrate the level of GCase in brain, spleen, liver andlung, respectively, of C57BL/6 mice administered a 2-week dosing regimenconsisting of nine doses of (i) control vehicle; (ii) 10 or 100 mg/kg(free base equivalent) of reference compound, IFG-tartrate; or (iii) 10or 100 mg/kg (free base equivalent) of test compound, Compound B. Notethat the values appearing above the bars in these figures represent thefold increase above control. Likewise, in these figures, the symbol “*”represents p<0.05 t-test compared to control and the symbol “#”represents p<0.05 t-test compared to mice treated with referencecompound, IFG-tartrate.

FIG. 6A illustrates the plasma level of test compound, Compound A, inrats administered a single intravenous dose of 3 mg/kg (free baseequivalent) of the hydrochloride salt form of Compound A.

FIG. 6B illustrates the plasma level of Compound A in rats administereda single p.o. dose of 10, 30, or 300 mg/kg (free base equivalent) of thehydrochloride salt form of Compound A.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the following terms shall have the definitions set forthbelow.

As used herein, the phrase “degenerative disorder of the central nervoussystem” means any disorder associated with the premature degeneration ofany component of the central nervous system, such as neurons, myelinsheaths or axons. Such disorders include but are not limited tomulti-infarct dementia, Huntington's disease, Pick's disease,amyotrophic lateral sclerosis, Creutzfeldt-Jakob's disease, frontal-lobedegeneration, corticobasal degeneration, progressive supranuclear palsy,Parkinson's disease, dementia with Lewy bodies, multiple system atrophyor Alzheimer's disease.

As used herein the term “treating” means to ameliorate one or moresymptoms associated with the referenced disorder.

As used herein, the term “preventing” means to mitigate a symptom of thereferenced disorder.

As used herein the phrase “an effective amount” means an amounteffective to prevent and/or treat a patient at risk for developing ordiagnosed with the referenced disorder, and thus producing the desiredtherapeutic effect.

As used herein the term “patient” means a mammal (e.g., a human).

Parkinson's disease may be diagnosed in patients according to the UnitedKingdom Parkinson's Disease Society brain-bank clinical diagnosticcriteria (see, Hughes et al., Accuracy of clinical diagnosis ofidiopathic Parkinson's disease: a clinico-pathological study of 100cases. J Neurol Neurosurg Psychiatry 1992; 55:181-184) and/or thecriteria described by Gelb et al., Diagnostic Criteria for Parkinson'sDisease. Arch Neurol. 1999; 56(1):33-39. Likewise, the severity ofParkinson's disease may be ascertained using the Unified Parkinson'sDisease Rating Scale. See, e.g., Fahn and Elton, Members of the UnifiedParkinson's Disease Rating Scale Development Committee. UnifiedParkinson's Disease Rating Scale. In: Fahn et al., Recent developmentsin Parkinson's disease. New York: Macmillan, 1987: 153-163.

Alzheimer's disease may be diagnosed in patients according to thecriteria for dementia of the Alzheimer's type of the Diagnostic andStatistical Manual of Mental Disorders, 4^(th) ed.: DSM-IV. Washington,D.C.: American Psychiatric Association, 1994. Likewise, the criteria forprobable Alzheimer's disease may be ascertained based on criteria of theNational Institute of Neurological and Communicative Disorders andStroke and the Alzheimer's Disease and Related Disorders Association.See also, McKhann et al., Clinical diagnosis of Alzheimer's disease:report of the NINCDS-ADRDA work group under the auspices of Departmentof Health and Human Services Task Force on Alzheimer's Disease.Neurology 1984; 34:939-944.

Multiple system atrophy (MSA) is characterized by glial cytoplasmicinclusion bodies (also known as Papp-Lantos bodies) in the movement,balance and automatic control centers of the brain. The most commonfirst sign of MSA is the appearance of an “akinetic-rigid syndrome”(i.e., slowness of initiation of movement resembling Parkinson'sdisease) found in 62% at first presentation. Other common signs at onsetinclude problems with balance (found in 22%), followed by genito-urinaryproblems (9%). For men, the first sign can be erectile dysfunction(unable to achieve or sustain an erection). Both men and women oftenexperience problems with their bladders including urgency, frequency,incomplete bladder emptying or an inability to pass urine (retention).About 1 in 5 MSA patients will suffer a fall in their first year ofdisease. As the disease progresses three groups of symptoms predominate.These are: (i) parkinsonism (slow, stiff movement, writing becomes smalland spidery); (ii) cerebellar dysfunction (difficulty coordinatingmovement and balance); and (iii) autonomic dysfunction (impairedautomatic body functions) including: postural or orthostatichypotension, resulting in dizziness or fainting upon standing up,urinary incontinence, impotence; constipation; dry mouth and skin;trouble regulating body temperature due to abnormal sweating; abnormalbreathing during sleep. Notably, not all of these symptoms areexperienced by all patients.

Dementia with Lewy bodies (DLB) is one of the most common types ofprogressive dementia. The central feature of DLB is progressivecognitive decline, combined with three additional defining features: (1)pronounced “fluctuations” in alertness and attention, such as frequentdrowsiness, lethargy, lengthy periods of time spent staring into space,or disorganized speech; (2) recurrent visual hallucinations, and (3)parkinsonian motor symptoms, such as rigidity and the loss ofspontaneous movement. People may also suffer from depression. Thesymptoms of DLB are caused by the build-up of Lewy bodies—accumulatedbits of alpha-synuclein protein—inside the nuclei of neurons in areas ofthe brain that control particular aspects of memory and motor control.Researchers don't know exactly why alpha-synuclein accumulates into Lewybodies or how Lewy bodies cause the symptoms of DLB, but they do knowthat alpha-synuclein accumulation is also linked to Parkinson's disease,multiple system atrophy, and several other disorders, which are referredto as the “synucleinopathies.” The similarity of symptoms between DLBand Parkinson's disease, and between DLB and Alzheimer's disease, canoften make it difficult for a doctor to make a definitive diagnosis. Inaddition, Lewy bodies are often also found in the brains of people withParkinson's and Alzheimer's diseases. These findings suggest that eitherDLB is related to these other causes of dementia or that an individualcan have both diseases at the same time. DLB usually occurssporadically, in people with no known family history of the disease.However, rare familial cases have occasionally been reported.

Chemical Syntheses a. Isofagomine (IFG;(3R,4R,5R)-5-(hydroxymethyl)-3,4-piperidinediol))

Isofagomine (IFG; (3R,4R,5R)-5-(hydroxymethyl)-3,4-piperidinediol))refers to a compound having the following structure:

Synthesis of isofagomine is described in U.S. Pat. Nos. 5,844,102 toSierks et al. at column 17, line 53 to column 20, line 6, incorporatedherein by reference and 5,863,903 to Lundgren et al. in Example 1, atcolumn 5, line 20 to column 7, line 33, incorporated herein byreference. Isofagomine tartrate, also known as IFG and Plicera™, hasbeen assigned CAS number 919364-56-0. Preparation of isofagominetartrate is described in US2007/0281975I at paragraphs [0046] to andpurification of isofagomine tartrate is described at paragraphs [0051]to [0053], both of which are specifically incorporated herein byreference.

b. Synthesis of (3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diolhydrochloride

(4aR,8R,8aR)-6-benzyl-2,2-dimethylhexahydro-4H-[1,3]dioxino[5,4,c]pyridin-8-ol(2)

To a solution of (3R,4R,5R-5-(hydroxymethyl)piperidine-3,4-diol (1) (5.9g, 40.0 mmol) in dry DMF (75 mL) was added K₂CO₃ (6.4 g, 46.0 mmol)followed by benzyl chloride (4.8 mL, 42.0 mmol) and the resultingmixture was heated to 70° C. for 14 hrs at which point the startingmaterial could not be detected by tlc. The solvent was evaporated invacuo and the residue was redissolved in a minimum amount of water. Thesolution was extracted 10-12× with EtOAc and the combined extracts weredried over Na₂SO₄ and filtered. The filtrate was evaporated in vacuo togive the title compound (7.5 g, 79%) as a tan solid which could be usedwithout further purification. The tan solid was dissolved in DMF (75 mL)and toluenesulfonic acid (6.7 g, 35.0 mmol) was added followed by2-methoxy propene (7.2 mL, 75.0 mmol) and the mixture was stirredovernight at room temperature. After stirring overnight an additional7.2 mL of 2-methoxy propene was added and the mixture was again stirredovernight at room temperature. At this point no starting material couldbe detected by tlc. Sodium hydroxide (50% aq, 5 mL) was added to thereaction mixture and the solvent was evaporated in vacuo. The residuewas dissolved in EtOAc and washed with water. The aqueous washes werecombined and washed 1× with EtOAc. This was combined with originalextracts, washed with brine and dried over MgSO₄. The solution wasfiltered and the solvent evaporated to give a waxy solid. This solid canbe directly used in the next step. An analytical sample can be obtainedfrom chromatography (0 to 5% (9/1 MeOH/NH₄OH) in CHCl₃) to obtain thetitle compound. ¹H NMR (CDCl₃) 1.35 (s, 3H), 1.39 (s, 3H), 1.66 (t, 1H),1.83 (m, 1H), 1.95 (t, 1H), 2.56 (ddd, 1H), 3.03 (ddd, 1H), 3.30 (t,1H), 3.6-3.8 (m, 5H), 7.27 (m, 5H).

(3R,4R,5R)-1-benzyl-3-(benzyloxy)-5-(hydroxymethyl)piperidin-4-ol (3)

To a solution of 2 (4.4 g, 15.9 mmol) in DMF (50 mL) was added 95% NaH(0.43 g, 17.9 mmol) and the resulting mixture was stirred 1 hr under N₂.Then benzyl chloride (1.9 mL, 16.3 mmol) was added and the mixture wasstirred overnight at room temperature. After approximately 14 hrs theDMF was evaporated in vacuo. The residue was dissolved in EtOAc andwashed with water and then brine and then dried over MgSO₄. The solutionwas filtered and the filtrate evaporated in vacuo to give the crudeproduct. Chromatography on silica gel eluting with 0-25% EtOAc/hexanegave 2.1 g of a white foam. ¹H NMR (CDCl₃) 1.15 (m, 1H), 1.19 (s, 3H),1.39 (s, 3H), 1.9-2.05 (m, 4H), 2.53 (ddd, 1H), 3.04 (ddd, 1H), 3.3 (s,1H), 3.45-3.8 (m, 4H), 7.2 (m, 10H). This was dissolved in MeOH (100 mL)and 1.5 mL of 6N HCl in 2-PrOH was added and the mixture was stirred atroom temperature. After 1 hr the starting material was gone as judged bytlc. The solvent was evaporated and the residue was dissolved in 10%Na₂CO₃ and extracted with EtOAc (2×). The combined extracts were washedwith one small portion of water and then brine and dried over MgSO₄. Thesolution was filtered and the filtrate was evaporated to give the titlecompound (1.7 g, 33%). ¹H NMR (CDCl₃) 1.6-1.95 (m, 3H), 2.63 (ddd, 1H),3.00 (ddd, 1H), 3.25-3.65 (m, 5H), 4.35 (d, 1H), 4.5 (d, 1H), 7.2 (m,10H).

((3R,4R,5R)-1-benzyl-4,5-bis(benzyloxy)-piperidin-3-yl)methanol (4b)

To a solution of 3 (1.1 g, 3.36 mmol) in DMF (10 mL), was added NaH(0.10 g, 4.0 mmol) and the reaction was stirred 30 minutes at roomtemperature at which point benzyl chloride (0.38 mL, 3.3 mmol) was addedand the reaction was stirred overnight at room temperature. The solventwas evaporated in vacuo and the residue was dissolved in EtOAc andwashed with water and then brine and dried over Na₂SO₄. The solution wasfiltered and the filtrate evaporated to give a mixture of 4a and 4b(roughly a 2/1 mixture). The mixture was chromatographed on silica geleluting with 0-50% EtOAc/hexane. The fractions corresponding to themajor regioisomer were combined to give 4a (0.49 g, 35%) as a colorlessoil. ¹H NMR (CDCl₃) 1.81 (t, 2H), 1.96 (m, 1H), 2.80 (m, 1H), 3.05 (ddd,1H), 3.3-3.6 (m, 6H), 4.41 (s, 2H), 4.55 (q, 2H), 7.2 (m, 15H). Thefractions of the minor regioisomer were combined to give 4b (0.23 g,16%) as a colorless oil. ¹H NMR (CDCl₃) 1.8-2.0 (m, 3H), 2.72 (dd, 1H),3.05 (ddd, 1H), 3.27 (t, 1H), 3.4-3.6 (m, 4H), 3.65 (m, 1H), 4.56 (s,2H), 4.60 (d, 1H), 4.90 (d, 1H), 7.2 (m, 15H).

(3R,4R,5R)-1-benzyl-3,4-bis(benzyloxy)-5-(fluoromethyl)piperidin-4-ol(5)

A solution of 4b (0.32 g, 0.77 mmol) in CH₂Cl₂ (40 mL) was cooled to −15to −20° C. in an ice/salt water bath. To this was added(diethylamino)sulfur trifluoride (0.15 mL, 1.15 mmol) and the reactionwas allowed to stir for about 20 minutes at which point the startingmaterial was consumed as judged by tlc. The reaction was quenched by theaddition of NaHCO₃ and the mixture was extracted with EtOAc (2×). Thecombined extracts were washed with brine and then dried over Na₂SO₄. Thesolution was filtered and the filtrate was evaporated to give the crudeproduct. Silica gel chromatography (0-25% EtOAc/hexane) afforded thedesired product (0.2 g, 63%) as a colorless oil. ¹H NMR (CDCl₃) 1.9-2.15(m, 3H), 2.88 (dd, 1H), 3.12 (ddd, 1H), 3.36 (t, 1H), 3.55 (d, 1H), 3.65(m, 1H), 4.3-4.6 (m, 3H), 4.63-4.65 (m, 3H), 4.95 (d, 1H), 7.2 (m, 15H).

(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol hydrochloride (6)

To a solution of 5 (0.24 g, 0.57 mmol) in EtOH (40 mL) was added 0.5 mLof 6N HCl in 2-PrOH. The solvent was evaporated in vacuo and thenco-evaporated 2× more with EtOH. The residue was dissolved in EtOH andhydrogenated at 55 psi using Pd(OH)₂ as catalyst. After 14 hrs thestarting material could no longer be detected and the solution wasfiltered and the filtrate was evaporated in vacuo. The residue wastriturated with acetone and then filtered to give an off white solid(0.09 g, 85%) as the title compound. ¹H NMR (DMSO-d₆) 1.95-2.05 (m, 1H),2.65 (t, 1H), 2.85 (t, 1H) 3.15-3.4 (m, 3H), 3.55 (m, 1H), 4.55 (m, 1H),4.65 (m, 1H), 5.45-5.6 (dd, 2H), 9.05 (br s, 2H).

(3R,4R,5R)-1-benzyl-3-(benzyloxy)-5-(fluoromethyl)piperidin-4-ol (7)

To a solution of 3 (26.0 g, 79.5 mmol) dissolved in pyridine (300 mL)and cooled in an ice bath was added TsCl (16.6 g, 87.5 mmol) inportions. After the addition was complete, the reaction mixture wasallowed to warm to room temperature and stirred overnight. The solventwas evaporated in vacuo and the residue dissolved in EtOAc. The solutionwas washed with water (2×) and then brine and then dried over MgSO₄. Thesolution was filtered and the filtrate evaporated in vacuo to give thecrude product which was dried under high vacuum. The residue wasdissolved in THF (400 mL) and 1.0 M Bu₄NF (100 mmol, 100 mL0 was addedand the mixture was heated at reflux. After 2 hrs, there was no startingmaterial remaining. The reaction mixture was diluted with EtOAc andwashed with water (2×) and then with brine and dried over MgSO₄. Thesolution was filtered and the filtrate was evaporated in vacuo. Thecrude product was purified by silica gel chromatography (25%EtOAc/hexane) to give the title compound (7.5 g, 29% for 2 steps).

(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol hydrochloride (6)

To a solution of 7 (7.5 g, 22.8 mmol) in EtOH (150 mL) was added 5N HClin 2-PrOH (7 mL). The solution was evaporated in vacuo, thenco-evaporated 2× more with EtOH. The resulting material was dissolved inEtOH (100 mL) and the resulting solution was hydrogenated overnight withPd(OH)₂ at 50 psi. The catalyst was removed by filtration and thefiltrate was evaporated in vacuo. The residue was triturated withacetone and a pale yellow solid was collected. The resulting solid wasrecrystallized from EtOH to give the title compound as an off-whitesolid mp 200-202° C. ¹H NMR DMSO d₆ consistent with previous spectra.

c. Synthesis of (3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol

One approach for obtaining the free base of(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol is to synthesize(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol hydrochloride asdescribed in section “b” above followed by chromatography of(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol hydrochloride using 5-15%MeOH/NH₄OH (9:1) in CHCl₃ which converts the HCl salt form of(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol to its free base.

d. Synthesis of (3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol

(2S,3S,4aR,8S,8aR)-6-benzyl-8-(chloromethyl)-2,3-dimethoxy-2,3-dimethyloctahydro-[1,4]dioxino[2,3-c]pyridine(2)

To a solution of((2S,3S,4aR,8R,8aR)-6-benzyl-2,3-dimethoxy-2,3-dimethyloctahydro-[1,4]dioxino[2,3-c]pyridine-8-yl)methanol(1) (2.5 g, 7.1 mmol) in chloroform (35 mL) was added thionyl chloride(1.3 mL, 18 mmol). The resulting mixture was heated at reflux for 4-8hours until reaction was judged complete by TLC (98:2 CH₂Cl₂/2-PrOH).Solvent and excess reagent were evaporated in vacuo and the residue waschromatographed (98:2 CH₂Cl₂/2-PrOH) to give the title compound (2.2 g,85%). Product was characterized via HPLC/MS (MH⁺=369). Purity was judgedas >95%.

(3R,4R,5S)-1-benzyl-5-(chloromethyl)piperidine-3,4-diol (3)

To a solution of(2S,3S,4aR,8S,8aR)-6-benzyl-8-(chloromethyl)-2,3-dimethoxy-2,3-dimethyloctahydro-[1,4]dioxino[2,3-c]pyridine(2) (2.2 g, 5.9 mmol) in dicholoromethane (15 mL) was addedtrifluoroacetic acid (0.35 mol, 26 mL). Reaction mixture was heated atreflux for about 1 hour at which point reaction was judged complete byTLC. Excess solvent and TFA were evaporated in vacuo and the residue waschromatographed using silica gel (2-8% MeOH in CHCl₃). Fractions werecombined and evaporated to give (1.3 g, 87%) as the title compound.Product was characterized via HPLC/MS (MH⁺=255) and judged to be >95%pure.

(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol (4)

5N HCl (4.7 mmol, 0.94 mL) was added to(3R,4R,5S)-1-benzyl-5-(chloromethyl)piperidine-3,4-diol (3) (5.1 mmol,1.3 g) dissolved in EtOH. Solution was evaporated in vacuo and thenco-evaporated 2-3× with EtOH. The residue was redissolved in EtOH andcombined with Pd(OH)₂ (0.27 g), and hydrogenated at 55 psi for 12 hrs.The catalyst was subsequently filtered through dicalite and the filtratewas evaporated to obtain crude (4) as an HCl salt. This was thenchromatographed (5-15% MeOH/NH₄OH (9:1) in CHCl₃) to give 0.4 g of (4)as a white solid with MH⁺ of 165 and ¹H NMR (DMSO-d₆) 1.6 (m, 1H), 2.2(m, 3H), 2.9 (m, 3H), 3.15 (m, 1H), 3.6 (m, 1H), 3.8 (dd, 1H), 4.68 (d,1H), 4.88 (d, 1H).

e. Synthesis of (3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diolhydrochloride

One approach for obtaining the hydrochloride salt of(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol is to synthesize(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol as described in section“d” above followed by reacidification with HCl (either as an aqueoussolution or in 2-PrOH as it is often sold) whereby the free base isconverted to the HCl salt form of(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol and subsequent isolationof (3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol hydrochloride.

Alternatively, the synthesis of(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol may be followed asdescribed in section “d” above with the exception of the lastchromatographic step whereby the HCl salt is chromatographed using 5-15%MeOH/NH₄OH (9:1) in CHCl₃ as this step results in the conversion of thecrude HCl salt form of (3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol tothe free base. Rather, the crude HCl salt form of(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol may be purified bycrystallization.

Salts, Solvates and Prodrugs

Compounds of the present invention include pharmaceutically acceptablesalts, solvates and pro-drugs of(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol (also referred to hereinas Compound A) and (3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol (alsoreferred to herein as Compound B). Pharmaceutically acceptable saltsinclude salts derived from inorganic bases such as Li, Na, K, Ca, Mg,Fe, Cu, Zn, Mn; salts of organic bases such asN,N′-diacetylethylenediamine, glucamine, triethylamine, choline,hydroxide, dicyclohexylamine, metformin, benzylamine, trialkylamine,thiamine; chiral bases like alkylphenylamine, glycinol, phenyl glycinol,salts of natural amino acids such as glycine, alanine, valine, leucine,isoleucine, norleucine, tyrosine, cystine, cysteine, methionine,proline, hydroxy proline, histidine, ornithine, lysine, arginine,serine; non-natural amino acids such as D-isomers or substituted aminoacids; guanidine, substituted guanidine wherein the substituents areselected from nitro, amino, alkyl, alkenyl, alkynyl, ammonium orsubstituted ammonium salts and aluminum salts. Salts may include acidaddition salts where appropriate which are, hydrochlorides, sulphates,nitrates, phosphates, perchlorates, borates, hydrohalides, acetates,tartrates, maleates, citrates, succinates, palmoates,methanesulphonates, benzoates, salicylates, benzenesulfonates,ascorbates, glycerophosphates, ketoglutarates. In one embodiment, thepharmaceutically acceptable salt of(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol is the hydrochloride salt(also referred to herein as Compound A-HCl).

“Solvate” denotes a physical association of a compound with one or moresolvent molecules. This physical association involves varying degrees ofionic and covalent bonding, including hydrogen bonding. In certaininstances the solvate will be capable of isolation, for example when oneor more solvent molecules are incorporated in the crystal lattice of thecrystalline solid. “Solvate” encompasses both solution-phase andisolatable solvates. “Hydrate” is a solvate wherein the solvent moleculeis H₂O. Other non-limiting examples of suitable solvates includealcohols (e.g., ethanolates, methanolates, and the like).

Prodrugs are compounds which are converted in vivo to active forms (see,e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design andDrug Action”, Academic Press, Chapter 8, incorporated herein byreference). Additionally, a discussion of prodrugs is provided in T.Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Volume 14 ofthe A.C.S. Symposium Series, and in Bioreversible Carriers in DrugDesign, Edward B. Roche, ed., American Pharmaceutical Association andPergamon Press, 1987, both of which are incorporated herein by referencethereto. Prodrugs can be used to alter the biodistribution (e.g., toallow compounds which would not typically enter the reactive site of theprotease) or the pharmacokinetics for a particular compound. Forexample, a carboxylic acid group, can be esterified, e.g., with a methylgroup or an ethyl group to yield an ester. When the ester isadministered to a subject, the ester is cleaved, enzymatically ornon-enzymatically, reductively, oxidatively, or hydrolytically, toreveal the anionic group. An anionic group can be esterified withmoieties (e.g., acyloxymethyl esters) which are cleaved to reveal anintermediate compound which subsequently decomposes to yield the activecompound.

Examples of prodrugs and their uses are well known in the art (See,e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19). The prodrugs can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound with a suitable derivatizing agent. For examplehydroxy groups can be converted into esters via treatment with acarboxilic acid in the presence of a catalyst. Examples of cleavablealcohol prodrug moieties include substituted and unsubstituted, branchedor unbranched lower alkyl ester moieties, (e.g., ethyl esters), loweralkenyl esters, di-lower alkyl-amino lower-alkyl esters (e.g.,dimethylaminoethyl ester), acylamino lower alkyl esters, acyloxy loweralkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenylester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g.,with methyl, halo, or methoxy substituents) aryl and aryl-lower alkylesters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxyamides.

All stereoisomers (for example, geometric isomers, optical isomers andthe like) of 5-(fluoromethyl)piperidine-3,4-diol and5-(chloromethyl)piperidine-3,4-diol (including those of the salts,solvates and prodrugs of these compounds as well as the salts andsolvates of the prodrugs), such as those which may exist due toasymmetric carbons on various substituents, including enantiomeric forms(which may exist even in the absence of asymmetric carbons), rotamericforms, atropisomers, and diastereomeric forms, are contemplated withinthe scope of this invention. Individual stereoisomers of these compoundsmay, for example, be substantially free of other isomers, or may beadmixed, for example, as racemates or with all other, or other selected,stereoisomers. The chiral centers of the aforementioned compounds canhave the S or R configuration as defined by the IUPAC 1974Recommendations. The use of the terms “salt”, “solvate” “prodrug” andthe like, is intended to equally apply to the salt, solvate and prodrugof enantiomers, stereoisomers, rotamers, tautomers, racemates orprodrugs of 5-(fluoromethyl)piperidine-3,4-diol and5-(chloromethyl)piperidine-3,4-diol.

Formulations

The therapeutic agent(s) can be formulated to be suitable for any routeof administration, including e.g., orally in the form of tablets orcapsules or liquid, or in sterile aqueous solution for injection. Whenthe therapeutic agent(s) is formulated for oral administration, tabletsor capsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinized maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well known in the art. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product for constitutionwith water or another suitable vehicle before use. Such liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); or preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The liquidpreparations may also contain buffer salts, flavoring, coloring orsweetening agents as appropriate. Preparations for oral administrationmay be suitably formulated to give controlled or sustained release ofthe therapeutic agent(s).

In certain embodiments of the present invention, the therapeuticagent(s) is administered in a dosage form that permits systemic uptake,such that the therapeutic agent(s) may cross the blood-brain barrier soas to exert effects on neuronal cells. For example, pharmaceuticalformulations of the therapeutic agent(s) suitable forparenteral/injectable use generally include sterile aqueous solutions(where water soluble), or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol,polyethylene glycol, and the like), suitable mixtures thereof, orvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be broughtabout by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, benzyl alcohol, sorbic acid, and thelike. In many cases, it will be reasonable to include isotonic agents,for example, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonosterate or gelatin.

Sterile injectable solutions are prepared by incorporating thetherapeutic agent(s) in the required amount in the appropriate solventwith various of the other ingredients enumerated above, as required,followed by filter or terminal sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

The formulation can contain an excipient. Pharmaceutically acceptableexcipients which may be included in the formulation are buffers such ascitrate buffer, phosphate buffer, acetate buffer, and bicarbonatebuffer, amino acids, urea, alcohols, ascorbic acid, phospholipids;proteins, such as serum albumin, collagen, and gelatin; salts such asEDTA or EGTA, and sodium chloride; liposomes; polyvinylpyrollidone;sugars, such as dextran, mannitol, sorbitol, and glycerol; propyleneglycol and polyethylene glycol (e.g., PEG-4000, PEG-6000); glycerol;glycine or other amino acids; and lipids. Buffer systems for use withthe formulations include citrate; acetate; bicarbonate; and phosphatebuffers. Phosphate buffer is a preferred embodiment.

The formulation can also contain a non-ionic detergent. Preferrednon-ionic detergents include Polysorbate 20, Polysorbate 80, TritonX-100, Triton X-114, Nonidet P-40, Octyl α-glucoside, Octyl β-glucoside,Brij 35, Pluronic, and Tween 20.

Routes of Administration

The therapeutic agent(s) may be administered orally or parenterally,including intravenously, subcutaneously, intra-arterially,intraperitoneally, ophthalmically, intramuscularly, buccally, rectally,vaginally, intraorbitally, intracerebrally, intradermally,intracranially, intraspinally, intraventricularly, intrathecally,intracisternally, intracapsularly, intrapulmonarily, intranasally,transmucosally, transdermally, or via inhalation. In one preferredembodiment, the therapeutic agent(s) is administered orally.

Administration of therapeutic agent(s) may be by periodic injections ofa bolus of the formulation, or may be administered by intravenous orintraperitoneal administration from a reservoir which is external (e.g.,an i.v. bag) or internal (e.g., a bioerodable implant). See, e.g., U.S.Pat. Nos. 4,407,957 and 5,798,113, each incorporated herein byreference. Intrapulmonary delivery methods and apparatus are described,for example, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, eachincorporated herein by reference. Other useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, pump delivery, encapsulated celldelivery, liposomal delivery, needle-delivered injection, needle-lessinjection, nebulizer, aerosolizer, electroporation, and transdermalpatch. Needle-less injector devices are described in U.S. Pat. Nos.5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications ofwhich are herein incorporated by reference. Any of the formulationsdescribed above can be administered using these methods.

Subcutaneous injections have the advantages allowingself-administration, while also resulting in a prolonged plasmahalf-life as compared to intravenous administration. Furthermore, avariety of devices designed for patient convenience, such as refillableinjection pens and needle-less injection devices, may be used with theformulations of the present invention as discussed herein.

Dosage

A suitable pharmaceutical preparation is in a unit dosage form. In suchform, the preparation is subdivided into suitably sized unit dosescontaining appropriate quantities of the active component, e.g., aneffective amount to achieve the desired purpose. In certain embodiments,the therapeutic agent(s) is administered in one or more daily doses(e.g., once-a-day, twice-a-day, thrice-a-day). In certain embodiments,the therapeutic agent(s) is administered in intermittently.

Exemplary dosing regimens are described in International patentapplication PCT/US08/61764 published as WO 2008/134628 on Jun. 11, 2008and U.S. patent application Ser. No. 12/604,855, filed on Oct. 23, 2009,both of which are incorporated by reference herein in their entirety. Inone embodiment, the therapeutic agent(s) is administered in anintermittent dosing regimen that includes an initial “loading dose”given daily, followed by a period of non-daily interval dosing.

The amount of effective therapeutic agent(s) for preventing or treatingthe referenced disorder can be determined on a case-by-case basis bythose skilled in the art. The amount and frequency of administration ofthe therapeutic agent(s) will be regulated according to the judgment ofthe attending clinician (physician) considering such factors as age,condition and size of the patient as well as risk for developingdisorder or severity of the symptoms of the referenced disorder beingtreated.

Combination Drug Therapy

The therapeutic agent(s) of the present invention can be administered incombination with at least one other therapeutic agent. Administration ofthe therapeutic agent(s) of the present invention with at least oneother therapeutic agent is understood to encompass administration thatis sequential or concurrent. In one embodiment, the therapeutic agentsare administered in separate dosage forms. In another embodiment, two ormore therapeutic agents are administered concurrently in the same dosageform.

In certain embodiments, the therapeutic agent(s) of the presentinvention are administered in combination with at least one othertherapeutic agent which is an anti-dyskinesia Agent (e.g., Carbidopa,Levodopa), an anti-infective agent (e.g., Miglustat), an antineoplasticagent (e.g., Busulfan, Cyclophosphamide), a gastrointestinal agent(e.g., Methylprednisolone), a micronutrient (e.g., Calcitriol,Cholecalciferol, Ergocalciferols, Vitamin D), a vasoconstrictor agent(e.g., Calcitriol).

In certain embodiments, the therapeutic agent(s) of the presentinvention are administered in combination with allopregnanolone, alow-cholesterol diet, or cholesterol-lowering agents such as statins(e.g., Lipitor®); fibrates such as fenofibrate (Lipidil®); niacin;and/or binding resins such as cholestyramine (Questran®).

In one embodiment, the therapeutic agent(s) of the present invention isadministered in combination with gene therapy. Gene therapy iscontemplated both with replacement genes such as glucocerebrosidase orwith inhibitory RNA (siRNA) for the SNCA gene. Gene therapy is describedin more detail in U.S. Pat. No. 7,446,098, filed on Feb. 17, 2004.

In one embodiment, the therapeutic agent(s) of the present invention isadministered in combination with at least one other therapeutic agentwhich is an anti-inflammatory agent (e.g., ibuprofen or other NSAID).

In one embodiment, the therapeutic agent(s) of the present invention isadministered in combination with a substrate inhibitor forglucocerebrosidase, such as N-butyl-deoxynojirimycin (Zavesca®;miglustat available from Actelion Pharmaceuticals, US, Inc., South SanFrancisco, Calif., US).

Combinations of the therapeutic agent(s) of the present invention withat least one other therapeutic agent which is a therapeutic agent forone or more other lysosomal enzymes are also contemplated. Following isa non-limiting list of therapeutic agents for lysosomal enzymes.

TABLE 1 LYSOSOMAL ENZYME THERAPEUTIC AGENT α-Glucosidase1-deoxynojirimycin (DNJ) GenBank Accession No. Y00839 α-homonojirimycincastanospermine Acid β-Glucosidase (β- isofagomine glucocerebrosidase)C-benzyl isofagomine and derivatives GenBank Accession No. J03059N-alkyl (C9-12)-DNJ Glucoimidazole (and derivatives) C-alkyl-IFG (andderivatives) N-alkyl-β-valeinamines Fluphenozine calystegines A₃, B₁, B₂and C₁ α-Galactosidase A 1-deoxygalactonojirimycin (DGJ) GenBankAccession No. NM000169 α-allo-homonojirimycin α-galacto-homonojirimycinβ-1-C-butyl-deoxynojirimycin calystegines A₂ and B₂ N-methylcalystegines A₂ and B₂ Acid β-Galactosidase 4-epi-isofagomine GenBankAccession No. M34423 1-deoxygalactonojirimyicn Galactocerebrosidase(Acid β- 4-epi-isofagomine Galactosidase) 1-deoxygalactonojirimycinGenBank Accession No. D25283 Acid α-Mannosidase 1-deoxymannojirimycinGenBank Accession No. U68567 Swainsonine Mannostatin A Acidβ-Mannosidase 2-hydroxy-isofagomine GenBank Accession No. U60337 Acidα-L-fucosidase 1-deoxyfuconojirimycin GenBank Accession No. NM_000147β-homofuconojirimycin 2,5-imino-1,2,5-trideoxy-L-glucitol2,5-deoxy-2,5-imino-D-fucitol 2,5-imino-1,2,5-trideoxy-D-altritolα-N-Acetylglucosaminidase 1,2-dideoxy-2-N-acetamido-nojirimycin GenBankAccession No. U40846 α-N-Acetylgalactosaminidase1,2-dideoxy-2-N-acetamido- GenBank Accession No. M62783galactonojirimycin β-Hexosaminidase A 2-N-acetylamino-isofagomineGenBank Accession No. NM_000520 1,2-dideoxy-2-acetamido-nojirimycinNagstatin β-Hexosaminidase B 2-N-acetamido-isofagomine GenBank AccessionNo. NM_000521 1,2-dideoxy-2-acetamido-nojirimycin Nagstatinα-L-Iduronidase 1-deoxyiduronojirimycin GenBank Accession No. NM_0002032-carboxy-3,4,5-trideoxypiperidine β-Glucuronidase 6-carboxy-isofagomineGenBank Accession No. NM_000181 2-carboxy-3,4,5-trideoxypiperidineSialidase 2,6-dideoxy-2,6, imino-sialic acid GenBank Accession No.U84246 Siastatin B Iduronate sulfatase 2,5-anhydromannitol-6-sulphateGenBank Accession No. AF_011889 Acid sphingomyelinase desipramine,phosphatidylinositol-4,5- GenBank Accession No. M59916 diphosphate

In certain embodiments, the therapeutic agent(s) of the presentinvention are administered in combination with at least one therapeuticagent which is an anti-dyskinesia Agent (e.g., Carbidopa, Levodopa), ananti-infective agent (e.g., Cyclosporine, Miglustat, Pyrimethamine), anantineoplastic agent (e.g., Alemtuzumab, Azathioprine, Busulfan,Clofarabine, Cyclophosphamide, Melphalan, Methotrexate, Rituximab), anantirheumatic agent (e.g., Rituximab) a gastrointestinal agent (e.g.,Methylprednisolone), a micronutrient (e.g., Calcitriol, Cholecalciferol,Ergocalciferols, Folic Acid, Vitamin D), a reproductive control agent(e.g., Methotrexate), a respiratory system agent (e.g.,Tetrahydrozoline), vasoconstrictor agent (e.g., Calcitriol,Tetrahydrozoline).

In certain embodiments, the therapeutic agent(s) of the presentinvention are administered in combination with at least one therapeuticagent which is a therapeutic agent for β-hexosaminidase A and/or atherapeutic agent for acid β-galactosidase. In certain embodiments, thetherapeutic agent(s) of the present invention are administered incombination with at least one therapeutic agent which is ananti-infective agent (e.g., Miglustat), an antineoplastic agent (e.g.,Alemtuzumab, Busulfan, Cyclophosphamide), a gastrointestinal agent(e.g., Methylprednisolone).

The therapeutic agent(s) of the present invention can be administered incombination with at least one other therapeutic agent which includes butis not limited to, RNAi, dopamine replacement (e.g., levadopa (L-DOPA)),dopamine replacement stabilizer (e.g., carbidopa, and entacapone),anticholinergic (e.g., trihexyphenidyl, benzotropine mesylate(Cogentin®), trihexyphenidyl HCL (Artane®), and procyclidine),catechol-O-methyltransferase (COMT) inhibitor (e.g., entacapone(Comtan®) and tolcapone (Tasmar®)), dopamine receptor agonist (e.g.,bromocriptine (Parlodel®), pramipexole (Mirapex®), ropinirole(Requip®)), pergolide (Permax), and APOKYN™ injection (apomorphinehydrochloride), monoamine oxidase (MAO) inhibitor (i.e., MAO-A and/orMAO-B inhibitors, e.g., selegiline (Deprenyl, Eldepryl®, Carbex®),selegiline HCl orally disintegrating tablet (Zelapar®), and rasagiline(Azilect®)), peripheral decarboxylase inhibitor, amantadine(Symmetrel®), and rivastigmine tartrate (Exelon®).

Also contemplated are combinations of the therapeutic agent(s) of thepresent invention with more than one other therapeutic agent. Exemplarycombinations of other therapeutic agents include, but not are notlimited to, carbidopa/levodopa (Sinemet® or Parcopa®), carbidopa,levodopa and entacapone (Stalevo®), levodopa with a dopamine receptoragonist such as bromocriptine (Parlodel®), pramipexole (Mirapex®),ropinirole (Requip®)), pergolide (Permax), or APOKYN™ injection(apomorphine hydrochloride).

In one embodiment, the therapeutic agent(s) of the present invention isadministered in combination with vaccine therapy, such as a vaccinecomprising alpha-synuclein and an adjuvant (Pilcher et al., LancetNeurol. 2005; 4(8):458-9).

In one embodiment, the therapeutic agent(s) of the present invention isadministered in combination with at least one other therapeutic agentthat may be protective such as dextromethorphan (Li et al., FASEB J.2005; April; 19(6):489-96); genistein (Wang et al., Neuroreport. 2005;February 28; 16(3):267-70), or minoclycline (Blum et al., Neurobiol Dis.2004; December; 17(3):359-66).

In one embodiment, the therapeutic agent(s) of the present invention isadministered in combination with at least one other therapeutic agentwhich is therapeutic agent for alpha-synuclein (e.g., Hsp70).

Patients having Parkinson's disease experience tremor, rigidity,bradykinesia, and postural imbalance. Patients having Lewy Body Dementiaexperience strong psychotic symptoms (visual hallucinations) in additionto mental decline such as memory loss and an inability to carry outsimple tasks. Observable improvements in symptoms, or a delay of onsetof certain symptoms in patients at risk of developing a disorder, or adelay in progression of the disorder will be evidence of a favorableresponse to the therapies provided herein.

In addition, measurable surrogate markers also may be useful forevaluating response to therapy. For instance, some investigators havereported detecting higher levels of alpha-synuclein or oligomeric formsof alpha-synuclein have been detected in the plasma of patients withParkinson's disease (Lee et al., J Neural Transm. 2006; 113(10):1435-9;El-Agnaf et al., FASEB J. 2006; 20(3):419-25), while some have reporteddecreased plasma alpha-synuclein in Parkinson's patients compared withnormal controls (Li et al., Exp Neurol. 2007; 204(2):583-8).

In certain embodiments, the therapeutic agent(s) of the presentinvention is administered in combination with at least one othertherapeutic agent which is an alcohol deterrent (e.g., Acamprosate), anarcotic analgesic (e.g., Remifentanil), an anti-dyskinesia agent (e.g,Amantadine, Apomorphine, Benserazide, Bromocriptine, Cabergoline,Carbidopa, Dexetimide, Droxidopa, Entacapone, Levodopa, Lisuride,Memantine, Piribedil, Prarnipexol, Ropinirole, Selegiline, Sinemet), ananti-infective agent (e.g, Amantadine, Amoxicillin, Clarithromycin,Ethanol, Interferons, Minocycline, PS-K), an anti-obesity agent (e.g.,Phenylpropanolamine, Topiramate), an anticonvulsant (e.g., Etiracetam,Topiramate), an antiemetic (e.g., Trimethobenzamide), anantihypertensive agent (e.g., Trandolapril), an antineoplastic agent(e.g., Cabergoline, PS-K), central nervous system depressant (e.g.,Aripiprazole, Benzocaine, Clozapine, Cocaine, Dexmedetomidine,Diphenhydramine, Isoflurane, Lithium, Lithium Carbonate, Methylperon,Morphine, Propofol, Quetiapine, Raclopride, Remifentanil, SodiumOxybate), a central nervous system stimulant (e.g., Caffeine citrate,Modafinil, Nicotine polacrilex), a coagulant (e.g., ArginineVasopressin, Deamino Arginine Vasopressin, Vasopressins), a dermatologicagent (e.g., Loratadine, Promethazine), a gastrointestinal agent (e.g.,Diphenhydramine, Domperidone, Omeprazole, Trimethobenzamide), a hypnoticand/or sedative (e.g., Remifentanil), a micronutrient (e.g.,Alpha-Tocopherol, Coenzyme Q10, Ergocalciferols, Hydroxocobalamin, Iron,Tocopherol acetate, Tocopherols, Vitamin B 12, Vitamin D, Vitamin E), aneuroprotective agent (e.g., Eliprodil, Modafinil, Rasagiline,Rivastigmine, Topiramate), a nootropic agent (e.g., Donepezil,Etiracetam), a psychotropic drug (e.g., Aripiprazole, Citalopram,Clozapine, Duloxetine, Lithium, Lithium Carbonate, Methylperon,Nortriptyline, Paroxetine, Quetiapine, Raclopride, Venlafaxine), arespiratory system agent (e.g., Dextromethorphan, Guaifenesin,Ipratropium, Naphazoline, Oxymetazoline, Phenylephrine,Phenylpropanolamine), a vasoconstrictor agent (e.g., Naphazoline,Oxymetazoline, Phenylephrine, Phenylpropanolamine).

In one preferred embodiment, the aforementioned other therapeutic agentsare administered when the disorder is Parkinson's disease.

In certain embodiments, the therapeutic agent(s) of the presentinvention is administered in combination with at least one othertherapeutic agent which is a nicotinic alpha-7 agonist (e.g., MEM 3454or MEM 63908 both of which are available from Memory Pharmaceuticals).In certain embodiments, the therapeutic agent(s) of the presentinvention is administered in combination with at least one othertherapeutic agent which is R3487 and/or R4996 (both of which areavailable from Roche). Also contemplated are combinations of thetherapeutic agent(s) of the present invention with more than one othertherapeutic agents. Exemplary combinations of other therapeutic agentsinclude, but not are not limited to, R3487/MEM 3454 and R4996/MEM 63908.

In certain embodiments, the therapeutic agent(s) of the presentinvention is administered in combination with at least onecholinesterase inhibitor (e.g., donepezil (brand name Aricept),galantamine (brand name Razadyne), and rivastigmine (branded as Exelonand Exelon Patch).

In certain embodiments, the therapeutic agent(s) of the presentinvention is administered in combination with at least onenoncompetitive NMDA receptor antagonist (e.g., memantine (brand namesAkatinol, Axura, Ebixa/Abixa, Memox and Namenda)).

In certain embodiments, the therapeutic agent(s) of the presentinvention is administered in combination with at least one othertherapeutic agent which is a non-narcotic analgesic (e.g., Celecoxib,Resveratrol, Rofecoxib, TNFR-Fc fusion protein), an anti-dyskinesiaagent (e.g., Dexetimide, Gabapentin, Levodopa, Memantine), ananti-infective agent (e.g., Acetylcysteine, Acyclovir, Benzoates,Deoxyglucose, Doxycycline, Interferon Alfa-2a, Interferon-alpha,Interferons, Moxifloxacin, PS-K, Quinacrine, Rifampin, Salicylic Acid,Valacyclovir), an anti-Inflammatory agent (e.g., Aspirin, Celecoxib,Curcumin, Ibuprofen, Indomethacin, Naproxen, Resveratrol, Rofecoxib,TNFR-Fc fusion protein), an anti-obesity agent (e.g.,Phenylpropanolamine), an anticonvulsant agent (e.g., Gabapentin,Homotaurine, Lamotrigine), an antiemetic (e.g., Olanzapine), anantihypertensive agent (e.g., Trandolapril), an antilipemic agent (e.g.,Atorvastatin, Choline, Clofibric Acid, Pravastatin, Simvastatin), anantineoplastic agent (e.g., Bryostatin 1, Carmustine, Cyclophosphamide,Interferon Alfa-2a, Leuprolide, Medroxyprogesterone 17-Acetate,Methyltestosterone, PK 11195, Prednisone, PS-K, Resveratrol,2,3-dihydro-1H-imidazo(1,2-b) pyrazole), an antirheumatic agent (e.g.,Aspirin, Celecoxib, Curcumin, Ibuprofen, Indomethacin, Naproxen,Resveratrol, Rofecoxib, TNFR-Fc fusion protein), a central nervoussystem depressant (e.g., Aripiprazole, Benzocaine, Cocaine, Gabapentin,Haloperidol, Haloperidol decanoate, Lithium, Lithium Carbonate,Lorazepam, Midazolam, Olanzapine, Perphenazine, Propofol, Quetiapine,Risperidone, Sodium Oxybate, Trazodone, Valproic Acid, Zolpidem), acentral nervous system stimulant (e.g., Caffeine citrate, Modafinil,Nicotine polacrilex), a channel blocker (e.g., Gabapentin, Lamotrigine),a coagulant (e.g., Antiplasmin, Vitamin K), a dermatologic agent (e.g.,Mineral Oil, Salicylic Acid), a gastrointestinal agent (e.g., Choline,Haloperidol, Lorazepam, Olanzapine, Omeprazole, TNFR-Fc fusion protein),a hypnotic and/or sedative agent (e.g., Zolpidem), a hypoglycemic agent(e.g., Insulin, Asp(B28)-, Rosiglitazone), a micronutrient (e.g.,Alpha-Tocopherol, Ascorbic Acid, Coenzyme Q10, Copper, Folic Acid,Hydroxocobalamin, Inositol, Iron, Niacin, Niacinamide, Nicotinic Acids,Pyridoxine, Selenium, Thioctic Acid, Tocopherol acetate, Tocopherols,Vitamin B 12, Vitamin B 6, Vitamin E, Vitamin K), a neuroprotectiveagent (e.g., Huperzine A, Modafinil, Nefiracetam, Rasagiline,Rivastigmine, (3-aminopropyl)(n-butyl)phosphinic acid), a nootropicagent (e.g., Donepezil, Nefiracetam), a platelet aggregation inhibitor(e.g., Resveratrol), a psychotropic drug (e.g., Aripiprazole, Bupropion,Citalopram, Duloxetine, Gabapentin, Haloperidol, Haloperidol decanoate,Lithium, Lithium Carbonate, Lorazepam, Midazolam, Nefiracetam,Olanzapine, Paroxetine, Perphenazine, Quetiapine, Risperidone,Sertraline, Trazodone, Tryptophan, Valproic Acid, Venlafaxine), areproductive control agent (e.g., Estradiol 17 beta-cypionate, Estradiol3-benzoate, Estradiol valerate, Indomethacin, Leuprolide,Medroxyprogesterone, Medroxyprogesterone 17-Acetate, Mifepristone), arespiratory system agent (e.g., Acetylcysteine, Dextromethorphan,Guaifenesin, Naphazoline, Oxymetazoline, Phenylephrine,Phenylpropanolamine), or a vasoconstrictor agent (e.g., Naphazoline,Oxymetazoline, Phenylephrine, Phenylpropanolamine).

In one preferred embodiment, the aforementioned other therapeutic agentsare administered when the disorder is Alzheimer's disease.

EXAMPLES

The present invention is further described by means of the examples,presented below. The use of such examples is illustrative only and in noway limits the scope and meaning of the invention or of any exemplifiedterm. Likewise, the invention is not limited to any particular preferredembodiments described herein. Indeed, many modifications and variationsof the invention will be apparent to those skilled in the art uponreading this specification. The invention is therefore to be limitedonly by the terms of the appended claims along with the full scope ofequivalents to which the claims are entitled.

Example 1 Determination of Inhibition Constants

The binding affinity (defined here by K_(i) binding constant) of GCasefor small molecule pharmacological chaperones described in this documentwere empirically determined using enzyme inhibition assays. In brief,the enzyme inhibition assays used monitored the ability of test compoundto bind and prevent the hydrolysis of a fluorogenic substrate in aconcentration-dependent manner. Specifically, the enzyme activity ofrecombinant human GCase (rhGCase; Cerezyme®, Genzyme Corp.) was measuredusing the 4-methylumbelliferyl-β-D-glucopyranoside (4-MU-β-D-Glc)fluorogenic substrate in the absence or in the presence of varyingamounts of each test compound. The resultant data were analyzed bycomparing all test samples to the no inhibition control sample (nocompound; corresponding to 100% enzyme activity) to determine theresidual enzyme activity in the presence of test compound. Thenormalized residual activity data were subsequently graphed (on y-axis)relative to the concentration of test compound (on x-axis) toextrapolate the test compound concentration which leads to 50%inhibition of enzyme activity (defined as IC₅₀). The IC₅₀ value for eachtest compound was then inserted into the Cheng-Prusoff equation(detailed below) to derive the absolute inhibition constant K_(i) thataccurately reflects the binding affinity of GCase for the test compound.The enzyme inhibition assays were performed at both pH 7.0 (endoplasmicreticulum pH) and at pH 5.2 (lysosomal pH) to gain insight into thebinding affinity (i.e., potency) of compounds for GCase in theendoplasmic reticulum and lysososome.

In Vitro Assay

Various concentrations of test compounds were prepared in buffer “M”consisting of 50 mM sodium phosphate buffer with 0.25% sodiumtaurocholate at pH 7.0 and pH 5.2. Enzyme (Cerezyme®, a recombinant formof the human enzyme β-glucocerebrosidase) was also diluted in the samebuffer “M” at pH 7.0 and pH 5.2. The substrate solution consisted of 3mM 4-methylumbelliferone β-D-glucopyranoside in buffer “M” with 0.15%Triton X-100 at both pH's. Five μl of diluted enzyme was added to 15 μlof the various inhibitor concentrations or buffer “M” alone andincubated at 37° C. for 1 hour with 50 μl of the substrate preparationto assess β-glucosidase activity at pH 7.0 and pH 5.2. Reactions werestopped by addition of an equal volume of 0.4 M glycine, pH 10.6.Fluorescence was measured on a plate reader for 1 sec/well using 355 nmexcitation and 460 nm emission. Incubations without added enzyme orwithout added inhibitors were used to define no enzyme activity andmaximum activity, respectively, and normalize % inhibition for a givenassay. The results of this inhibition assay for reference compound,IFG-tartrate, and test compounds, the hydrochloride salt form ofCompound A and Compound B, are summarized below in Table 2A.

TABLE 2A In vitro Determination of Inhibition Constants IC₅₀ (mM) Ki(μM) IC₅₀ (μM) Ki (μM) Compound pH 5.2 pH 5.2 pH 7 pH 7 IFG-Tartrate0.0437 ± 0.0018 0.023 ± 0.001  0.00626 ± 0.00018  0.0033 ± 0.00013Compound A- 0.014 ± 0.006 0.006 ± 0.003  0.006 ± 0.0005 0.003 ± 0.0003HCl Compound A- 0.014 ± 0.001 0.006 ± 0.0004 0.007 ± 0.0004 0.004 ±0.0002 HCl* Compound B 0.0729 ± 0.004  0.384 ± 0.0021 0.0098 ± 0.0005 0.0051 ± 0.0003  Note: *independent synthesis of the hydrochloride saltform of Compound AIn Situ Assay

The effect of IFG and its derivatives on lysosomal GCase activity wasassayed in situ using fibroblasts established from a normal subject.Cells seeded in 48-well plates were incubated with the indicatedconcentrations of compound for 16-24 hours. For the dose-responseassays, cells were incubated with the in situ substrate5-(pentafluorobenzoylamino)fluorescein di-β-D-glucopyranoside(PFBFDβGlu) for 1 hour and subsequently lysed to determine the extent ofsubstrate hydrolysis in the presence of compound. The assay employed arange of 12 concentrations encompassing 5 orders of magnitude, centeredon the IC50. Specifically, the following concentration ranges wereemployed: Compound A: 1×10⁻⁵ to 3.33×10⁻¹¹; IFG: 3.33×10⁻⁵ to 1×10⁻¹⁰;Compound B: 3×10⁻⁵ to 9×10⁻¹¹; wherein compound was serially diluted 1:3from the highest concentration in the ranges specified. Inhibition wasdetermined as the ratio of activity in the presence of compound to thatin the absence of compound. For the washout assays, cells were treatedwith compound for 16-24 hours at a concentration equal to the IC90.Cells were washed extensively and incubated in drug-free medium to allownet compound efflux from cells. Cells were then tested for lysosomalGCase activity at 2 hour intervals over a total period of 8 hoursfollowing compound removal. The increase in activity over time wasfitted with a single exponential function to determine the compound'swashout time. The results of these inhibition assays are summarizedbelow in Table 2B.

TABLE 2B In situ Determination of Inhibition Constants In Situ IC₅₀ InSitu EC₅₀ Emax Compound (μM) Washout (hr) (μM) (%) IFG-Tartrate 0.271 ±0.012 8.2 ± 0.04 0.9 ± 0.2 93.6 ± 2.5 Compound A-HCl 0.017 ± 0.008 6.6 ±0.12 0.00138 ± 0.00063 96.8 ± 7.7 Compound A-HCl* 0.0056 ± 0.0015   104± 4.58 Compound B 0.121 ± 0.007 2.9 ± 0.22 0.0096 ± 0.001  104.2 ± 3.4 Notes: *independent synthesis of the hydrochloride salt form of CompoundA Cheng-Prusoff equation: Ki = IC₅₀ /(1 + [S]/K_(m)) where [S] =substrate concentration; 2.5 mM 4-MU-β-D-Glc was used K_(m) = Michaelisconstant that defines substrate affinity; 1.8 ± 0.6 mM for 4-MU-β-D-Glc(Liou et al., (2006) J Biol. Chem. 281 (7), 4242-53)

Notably, test compounds, the hydrochloride salt form of Compound A andCompound B, were found to cause a concentration-dependent increase inGCase activity. Moreover, when compared to IFG-tartrate, test compounds,the hydrochloride salt form of Compound A and Compound B, enhancedenzyme activity to the same maximum level at much lower concentration.

Example 2 Blood Brain Barrier Penetration

The blood-brain barrier (BBB) penetration of reference compound(IFG-tartrate) and test compounds (IFG-derivatives, the hydrochloridesalt form of Compound A and Compound B) were assayed after oraladministration to mice. For this purpose, 8-week old wild-type male mice(C57BL/6) were administered a single 30 mg/kg (free base equivalent)p.o. dose of reference or test compound by gavage (n=3 mice per timepoint). Dosing solutions were prepared in water. After dosing, mice wereeuthanized with CO₂ at the following time points: 0-, 0.5-, 1-, and4-hour post-dose. After euthanization, whole blood was collected fromthe inferior vena cava into lithium heparin tubes. Similarly, brainswere collected from each mouse. Plasma was derived by spinning wholeblood at 2700 g for 10 minutes at 4° C. followed by storage on dry ice.Whole brains were washed in cold PBS to remove contaminating blood,blotted dry, flash frozen on dry ice, and ultimately stored at −80° C.until analysis. To prepare brain samples for analysis, 50-100 mg oftissue was homogenized in 400 μl of water/mg tissue. Samples were thenclarified by centrifugation. Next, 25 μl of the brain homogenatesupernatant or 25 μl of plasma were combined with 25 μl ofacetonitrile:water (95/5). This was supplemented with 25 μl ofacetonitrile and 50 μl of internal standard (100 ng/mL IFG 13C2-15N in0.5% formic acid in (70:30) acetonitrile:methanol. Samples were againclarified by centrifugation and 75 μl of the supernatant was combinedwith 75 μl of acetonitrile. Samples were then analyzed for compoundlevels by LC-MS/MS at PPD Inc. (3230 Deming Way, Middleton, Wis. 53562).In brief, a Thermo Betasil, Silica-100, 50×3 mm, 5μ column equilibratedwith a mixture of mobile phase consisting of 5 mM ammonium formate and0.05% formic acid in (A) 95:5 acetonitrile:water or (B) 70:20:10methanol:water:acetonitrile was employed. Between 20 and 30 μl samplewas injected for analysis. Notably, the retention times for IFG,Compound A and Compound B are 4.91, 4.33, and 4.32 minutes,respectively. For MS/MS, the analytes were monitored by MRM with thefollowing ion masses (Q1/Q3, amu): IFG 13C2-15N isotopically-labeledinternal standard (151.1/115.1), IFG (148.1/112.1), Compound A(150.1/103.1), Compound B (166.2/130.1). When Compound B was analyzed,Compound A was used as internal standard (150.1/130.1). For calculatingdrug concentrations, raw data for plasma (ng/mL) and brain (ng/g) wasconverted to nM using the molecular weight of respective compounds andassuming 1 g of tissue is equivalent to 1 mL volume. Concentration as afunction of time was plotted in GraphPad Prism version 4.02.

The plasma levels and brain levels of reference compound, IFG, and testcompound, Compound A, detected in mice administered a single 30 mg/kg(free base equivalent) p.o. dose of IFG-tartrate or the hydrochloridesalt form of Compound A are illustrated in FIGS. 1A and 1B,respectively. Similarly, the ratio of brain to plasma levels of CompoundA and IFG detected in these mice is illustrated in FIG. 1C.Surprisingly, these results reflect that Compound A crossed the bloodbrain barrier more readily as compared to IFG.

The brain levels of reference compound, IFG, and test compound, CompoundB, detected in mice administered a single 30 mg/kg (free baseequivalent) p.o. dose of IFG-tartrate or free base form of Compound B isillustrated in FIG. 2. Notably, even higher levels of Compound B weredetected in brain than that observed following administration of thehydrochloride salt form of Compound A.

Example 3 GCase Enhancement

The ability of orally administered test compounds (the hydrochloridesalt form of Compound A and Compound B) to elevate GCase levels ascompared to reference compound, IFG-tartrate, was assessed in mice. Forthis purpose, 8-week old wild-type male mice (C57/BL6) were administereda single p.o. (gavage) dose (detailed in FIGS. 3A-D, 4A-D, and 5A-D) ofcontrol, reference compound (IFG-tartrate) or test compound (thehydrochloride salt form of Compound A or Compound B). Seven animals wereused per dose. The dosing solutions were prepared in water. Compoundswere administered over 2 weeks as follows: week 1, Mon-Fri (On), Sat-Sun(Off); week 2, Mon-Thu (On); necropsy on Friday. Thus, a total of 9doses (dosing solutions prepared fresh every day) were given to eachmouse, with a 24-hour washout between the last dose and necropsy.

After completion of dosing, mice were euthanized with CO₂ and wholeblood was drawn into lithium heparin tubes from the inferior vena cava.Plasma was collected by spinning blood at 2700 g for 10 minutes at 4° C.Liver, spleen, lung, and brain tissues were removed, washed in cold PBS,blotted dry, flash frozen on dry ice, and stored at −80° C. untilanalysis. GCase levels were measured by homogenizing approximately 50 mgtissue in 500 μl McIlvane (MI) buffer (100 mM sodium citrate, 200 mMsodium phosphate dibasic, 0.25% sodium taurocholate, and 0.1% TritonX-100, pH 5.2) at pH 5.2 for 3-5 seconds on ice with a microhomogenizer. Homogenates were then incubated at room temperature withoutand with 2.5 mM conduritol-B-epoxide (CBE) for 30 min. Finally, 3.7 mM4-methylumbeliferryl-β-glucoside (4-MUG) substrate was added andincubated at 37° C. for 60 min. Reactions were stopped by addition of0.4 M glycine, pH 10.6. Fluorescence was measured on a plate reader for1 sec/well using 355 nm excitation and 460 nm emission. Total proteinwas determined in lysates using the MicroBCA kit according to themanufacturer's instructions. A 4-methylumbelliferone (4-MU) standardcurve ranging from 1.0 nM to 50 μM was run in parallel for conversion ofraw fluorescence data to absolute GCase activity (in the presence andabsence of CBE) and expressed as nanomoles of 4-MU released permilligram of protein per hour (nmol/mg protein/hr). GCase levels andprotein levels were calculated using Microsoft Excel (Redmond, Wash.)and GraphPad Prism version 4.02.

FIGS. 3A-D illustrate the level of GCase in brain, spleen, liver andlung, respectively, of C57BL/6 mice administered a 2-week dosing regimenconsisting of nine doses of (i) control vehicle; (ii) 100 mg/kg (freebase equivalent) of reference compound, IFG-tartrate; or (iii) 10 or 100mg/kg (free base equivalent) of test compound, the hydrochloride saltform of Compound A. Furthermore, Tables 3A-C detail the level of GCaseenhancement in brain, spleen, and plasma, respectively, of mice treatedas described above.

TABLE 3A Brain samples of GCase enhancement study GCase IC50 Dose Dosingincrease Concentration pH 5.2 Compound (mg/kg) solution (-fold) (nM)(nM) Compound A 10 water 2.5 124 ± 11 (0.5) 10 ± 1 Compound A 100 water3.5 726 ± 51 (3) IFG 100 water 2.2 182 ± 13 (0.7) 50 ± 3 Notes: BLQ < 5ng/g Values in parenthesis indicate concentrations in GCase assays afterdilution IC50 data is average of three independent experiments

TABLE 3B Spleen samples of GCase enhancement study GCase IC50 DoseDosing Increase Concentration pH 5.2 Compound (mg/kg) solution (-fold)(nM) (nM) Compound A 10 water 1.9 69 ± 6 (0.3) 10 ± 1 Compound A 100water 2.8 304 ± 63 (1.1) IFG 100 water 1.9 711 ± 100 (2.6) 50 ± 3 Notes:BLQ < 8 ng/g Values in parenthesis indicate concentrations in GCaseassays after dilution IC50 data is average of three independentexperiments

TABLE 3C Plasma samples of GCase enhancement study Dose DosingConcentration Compound (mg/kg) solution (nM) Compound A 10 water 32 ±2.4 Compound A 100 water 254 ± 73.5 IFG 100 water 330 ± 36   Note: BLQ <1 ng/mL

Similarly, FIGS. 4A-D illustrate the level of GCase in brain, spleen,liver and lung, respectively, of C57BL/6 mice administered a 2-weekdosing regimen consisting of nine doses of (i) control vehicle; (ii) 100mg/kg (free base equivalent) of reference compound, IFG-tartrate; or(ii) 1, 3, 10, 30 or 100 mg/kg (free base equivalent) of test compound,the hydrochloride salt form of Compound A.

As reflected in FIGS. 3A-D and 4A-D as well as Tables 3A-3C, miceadministered the hydrochloride salt form of Compound A demonstratedstatistically significant greater GCase enhancement in brain, spleen,liver and lung as compared to mice administered control or IFG-tartrate.Moreover, GCase enhancement in mice administered the hydrochloride saltform of Compound A was unexpectedly greater than that observed withIFG-tartrate even when far lower doses of the hydrochloride salt form ofCompound A were administered than IFG-tartrate.

Likewise, FIGS. 5A-D illustrate the level of GCase detected in brain,spleen, liver, and lung, respectively, of mice treated with Compound Band IFG-tartrate as described above. Furthermore, Tables 4A-4C detailthe level of GCase enhancement in brain, spleen, and plasma,respectively, of mice treated as described above.

TABLE 4A Brain samples of GCase enhancement study GCase IC50 Dose DosingIncrease Concentration pH 5.2 Compound (mg/kg) solution (-fold) (nM)(nM) Compound B 10 water 1.3 246 ± 42 (0.9) 73 ± 9 Compound B 100 water3.5 2066 ± 231 (7.6) IFG 10 water 1.5 49 ± 2.4 (0.2) 50 ± 3 IFG 100water 2.1 287 ± 74 (1.1) Notes: BLQ < 5 ng/g Values in parenthesisindicate concentrations in GCase assays after dilution IC50 data isaverage of three independent experiments

TABLE 4B Spleen samples of GCase enhancement study GCase IC50 DoseDosing Increase Concentration pH 5.2 Compound (mg/kg) solution (-fold)(nM) (nM) Compound B 10 water 1.4 645 ± 214 (2.4) 73 ± 9 Compound B 100water 2.2 2156 ± 464 (8) IFG 10 water 1.6 150 ± 12 (0.5) 50 ± 3 IFG 100water 2.0 1139 ± 220 (4.2) Notes: BLQ < 5 ng/g Values in parenthesisindicate concentrations in GCase assays after dilution IC50 data isaverage of three independent experiments

TABLE 4C Plasma samples of GCase enhancement study Dose DosingConcentration Compound (mg/kg) solution (nM) Compound B 10 water 104.3 ±15   Compound B 100 water 1529 ± 601  IFG 10 water 67 ± 10 IFG 100 water852 ± 134 Note: BLQ < 1 ng/mL

As reflected in FIGS. 5A-D as well as Tables 4A-4C, mice administeredCompound B demonstrated statistically significant greater GCaseenhancement in brain, spleen, liver and lung as compared to miceadministered control or IFG-tartrate. Moreover, GCase enhancement inmice administered Compound B was unexpectedly greater than that observedwith IFG-tartrate even when far lower doses of Compound B wereadministered than IFG-tartrate.

Example 4 Rat Pharmacokinetics

Pharmacokinetic (PK) data was obtained in rats to assess thebioavailability of test compound. In particular, the following PKparameters were calculated: bioavailability as measured by area underthe Concentration/Time curve (AUC), fraction of dose available (% F;further defined below), clearance (CL), volume of distribution (Vd), andhalf-life (t½). For this purpose, 8-week old Sprague-Dawley male ratswere given either a single intravenous (IV) dose equivalent to 3 mg/kgof free base or single escalating p.o. (gavage) doses of test compoundequivalent to 10, 30, and 100 mg/kg of free base. Three rats were usedper dosing group. Blood was collected over a 24-hr period. The timepoints for blood collection after intravenous administration were: 0,2.5, 5, 10, 15, 30, 45 min, 1, 2, 4, 8, 12, and 24 hrs; time points forblood collection after p.o. administrations were: 0, 5, 15, 30, 45 min,1, 2, 3, 4, 8, 12, and 24 hrs. Plasma samples were analyzed for compoundlevels by LC-MS/MS at PPD. Raw data was analyzed by non-compartmentalanalysis in Win-nonLin to calculate Vd, % F, CL, and t½.

Plasma levels in rats following a single 3 mg/kg (free base equivalent)intravenous dose of the hydrochloride salt form of Compound A areillustrated in FIG. 6A. Similarly, plasma levels in rats administered asingle dose of 10, 30, and 300 mg/kg (free base equivalent) p.o. dose ofthe hydrochloride salt form of Compound A are illustrated in FIG. 6B.Various pharmacokinetic parameters for Compound A based on theaforementioned study are detailed in Table 5 below.

TABLE 5 Rat PK data for Compound A Dose (mg/kg) Free AUC_(last) t1/2Cmax CL V_(D) Salt Base Route (hr*ng/mL) % F (h) (ng/mL) (mL/hr/kg)(mL/kg) 3.72 3 IV  1798 ± 42.7 N/A 1.4 ± 0.1 7274 ± 139 1666 ± 38.5 3481 ± 232 12.4 10 PO  2982 ± 228 50 ± 3.8 1.2 ± 0.4 1289 ± 271 3387 ±277 13283 ± 1929 37.2 30 PO  8251 ± 388 46 ± 2.5 2.3 ± 0.3 2870 ± 3554009 ± 509 13532 ± 3291 124 100 PO 29606 ± 1748 49 ± 2.7 2.6 ± 0.1 9393± 742 3393 ± 209 13101 ± 1518 Notes: Non compartmental analysis meanvalues ( N = 3 rats) BLD Below Limit of Detection (<0.5 ng/mL) BLQ BelowLimit of Quantitation${\%{\mspace{11mu}\;}F} = \frac{{AUC}\mspace{14mu}{PO} \times 100\mspace{14mu}{dose}\mspace{14mu}{normalized}}{{AUC}\mspace{14mu}{IV}}$AUC_(last) = Area under the Concentration/Time curve to the last datapoint

As reflected in FIGS. 6A and 6B as well as Table 5, the hydrochloridesalt form of Compound A has a favorable pharmacokinetic profile for drugdevelopment as a pharmacological chaperone. In particular, thehydrochloride salt form of Compound A shows good oral bioavailability(˜50%) and dose proportionality, a half-life of 1.0 to 2.5 hours, and avolume of distribution suggesting adequate penetration into peripheraltissues.

1. A method for treating a degenerative disorder of the central nervoussystem in a patient at risk for developing or diagnosed with the same,which comprises administering to the patient in need thereof aneffective amount of a therapeutic agent which is5-(fluoromethyl)piperidine-3,4-diol,5-(chloromethyl)piperidine-3,4-diol, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof, or any combination of two or morethereof.
 2. The method of claim 1, which comprises administering5-(fluoromethyl)piperidine-3,4-diol or a pharmaceutically acceptablesalt, solvate, or prodrug thereof.
 3. The method of claim 1, whichcomprises administering (3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diolor a pharmaceutically acceptable salt, solvate, or prodrug thereof. 4.The method of claim 1, which comprises administering(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol hydrochloride.
 5. Themethod of claim 1, which comprises administering5-(chloromethyl)piperidine-3,4-diol or a pharmaceutically acceptablesalt, solvate, or prodrug thereof.
 6. The method of claim 1, whichcomprises administering (3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diolor a pharmaceutically acceptable salt, solvate, or prodrug thereof. 7.The method of claim 1, which comprises administering(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol.
 8. The method of claim1, wherein the degenerative disorder is a synucleinopathy.
 9. The methodof claim 1, wherein the degenerative disorder is characterized by Lewybodies.
 10. The method of claim 1, wherein the degenerative disorder isParkinson's disease, dementia with Lewy bodies, multiple system atrophyor Alzheimer's disease.
 11. The method of claim 1, wherein thedegenerative disorder is associated with aggregation of at least oneprotein.
 12. The method of claim 1, wherein the degenerative disorder isassociated with aggregation of alpha-synuclein.
 13. The method of claim1, wherein the degenerative disorder is associated with aggregation ofnon-Abeta component.
 14. The method of claim 1, wherein the degenerativedisorder is associated with accumulation of at least one glycolipid. 15.The method of claim 1, wherein the degenerative disorder is associatedwith accumulation of at least one glycosphingolipid.
 16. The method ofclaim 1, wherein the degenerative disorder is associated withaccumulation of glucocerebroside.
 17. The method of claim 1, wherein thedegenerative disorder is associated with a mutation inglucocerebrosidase.
 18. The method of claim 1, further comprisingadministering an effective amount of at least one other therapeuticagent.
 19. The method of claim 18, wherein at least one othertherapeutic agent is levodopa, an anticholinergic, a catechol-O-methyltransferase inhibitor, a dopamine receptor agonist, a monoamine oxidaseinhibitor, a peripheral decarboxylase inhibitor, or an anti-inflammatoryagent.
 20. A method for treating Parkinson's disease in a patient atrisk for developing or diagnosed with the same, which comprisesadministering to the patient in need thereof an effective amount of(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol,(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof, or any combination of twoor more thereof.
 21. The method of claim 20, which comprisesadministering (3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol or apharmaceutically acceptable salt, solvate, or prodrug thereof.
 22. Themethod of claim 20, which comprises administering(3R,4R,5R)-5-(fluoromethyl)piperidine-3,4-diol hydrochloride.
 23. Themethod of claim 20, which comprises administering(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.
 24. The method of claim20, which comprises administering(3R,4R,5S)-5-(chloromethyl)piperidine-3,4-diol.
 25. The method of claim20, further comprising administering an effective amount of at least oneother therapeutic agent.
 26. The method of claim 25, wherein at leastone other therapeutic agent is levodopa, an anticholinergic, acatechol-O-methyl transferase inhibitor, a dopamine receptor agonist, amonoamine oxidase inhibitor, a peripheral decarboxylase inhibitor, or ananti-inflammatory agent.